Expression Of GUS Reporter Gene Research Articles

Bioinformatics analyses of rice calmodulin-lysine N-methyltransferases gene (OsCaM KMT) and characterization of its promoter from Oryza sativa L. sub sp. indica through transgenic approach

• Cloning of OsCaM KMT gene and promoter region from “Swarna”, an indica rice variety. • Presence of OsCAM KMT transcript in meristematic zone of rice plants, with higher expression in root tips suggesting they might be involved in auxin responsiveness. • Promoter analysis of OsCaM KMT::GUS construct depicted strong expression of GUS reporter gene in root tip, lateral roots and young leaf tip region. • Sequencing result indicated single nucleotide difference between the OsCAM KMT CDS from indica and japonica rice variety, resulting in silent mutation. Calmodulin-lysine N-methyltransferases (CaM KMT) belong to a family of protein lysine methyltransferases that catalyzes methylation of non-histone proteins. Lysine present at 116 position in the protein sequence of calmodulin is apparently methylated by CaM KMT in most species, however not characterized in rice. In the present study rice calmodulin-lysine N-methyltransferase ( OsCaM KMT ) gene has been isolated from an indica rice variety ‘Swarna’. Derived amino acid sequences of CaM KMT gene from rice and other plants were used in constructing a phylogenetic tree to show the evolutionary relationship among those species. Motif analysis along with comparative protein modeling was done to find out structural relationships among rice, Arabidopsis, and human CaM KMT. Gene expression analysis revealed differential tissue specific expression of OsCaM KMT . The highest transcript expression was revealed in the root tip followed by flower and new leaf, while, the least expression was found in the old leaf. In silico analysis was performed considering the 2 kilobases (kb) upstream promoter region of OsCaM KMT gene to find out various transcription factor binding sites. Analyses of the putative cis-regulatory elements revealed their involvement in tissue specific and stress dependent responses. For further characterization, the promoter region (2 kb) of OsCaM KMT gene was isolated, cloned, and sequenced. Histochemical GUS detection of transgenic rice depicted strong OsCaM KMT promoter expression in midribs, stomata, and tip of leaf tissues, central cylinder as well as tip parts of the primary root and lateral roots, along with significant expression in pollen grains, ovary, and vascular bundles. OsCaM KMT gene expression was corroborated by the histological GUS detection in OsCaM KMT promoter:: GUS fusion construct. Although several putative microRNAs (miRNAs) has been identified targeting OsCaM KMT coding DNA sequence (CDS) and UTR region, further studies are required to elucidate their tissue-specific regulation.

The root-specific NtR12 promoter-based expression of RIP increased the resistance against bacterial wilt disease in tobacco.

Tobacco is an important economic crop, but the quality and yield have been severely impaired by bacterial wilt disease (BWD) caused by Ralstonia solanacearum. Here, we describe a transgenic approach to prevent BWD in tobacco plants. A new root-specific promoter of an NtR12 gene was successfully cloned. The NtR12 promoter drove GUS reporter gene expression to a high level in roots but to less extent in stems, and no significant expression was detected in leaves. The Ribosome-inactivating proteins (RIP) gene from Momordica charantia was also cloned, and its ability to inhibit Ralstonia solanacearum was evaluated using RIP protein produced by the prokaryotic expression system. The RIP gene was constructed downstream of the NtR12 promoter and transformed into the tobacco cultivar "Cuibi No. 1" (CB-1), resulting in many descendants. The resistance against BWD was significantly improved in transgenic tobacco lines expressing NtR12::RIP. This study confirms that the RIP gene confers resistance to BWD and the NtR12 as a new promoter for its specific expression in root and stem. Our findings pave a novel avenue for transgenic engineering to prevent the harmful impact of diseases and pests in roots and stems.

Enhancing plant growth and biomass production by overexpression of GA20ox gene under control of a root preferential promoter.

Overexpression of GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameter, etc. To avoid these effects, we identified and employed different tissue-specific promoters for GA20ox overexpression. In this study, we examined the utility of At1g promoter to drive the expression of GUS (β-glucuronidase) reporter and AtGA20ox genes in tobacco and Melia azedarach. Histochemical GUS assays and quantitative real-time-PCR results in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of AtGA20ox gene under the control of At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants. Stem length as well as stem and root fresh weight increased by up to 1.5-3 folds in transgenic tobacco and 2 folds in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in root xylem width with xylem to phloem ratio over 150-200% as compared to WT plants. Importantly, no significant difference in leaf shape and size was observed between At1g::AtGA20ox transgenic and WT plants. These results demonstrate the great utility of At1g promoter, when driving AtGA20ox gene, for growth and biomass improvements in woody plants and potentially some other plant species.

Characterization of Protease Inhibitor Promoter from Black Gram (Vigna mungo L.) and gus Gene Expression in Transgenic Tobacco Plants

Like other legumes, black gram is also known to be source of plant protease inhibitors. Plant protease inhibitors (PIs) are small proteins which represent a class of well studied compounds of plant defense genes and are abundant in storage organs such as seeds and tubers. The protease inhibitor proteins serve various functions in the growth and developmental processes. Like other legumes black gram (Vigna mungo L.) is also known to be a source of plant protease inhibitors and considered to be involved in insect tolerance. In this study, promoter sequence consisting of 0.75 fragments of a 52 upstream flanking region preceding a protease inhibitor gene of black gram was cloned from genomic clones and its sequence is characterized. The spatial and temporal control of the 0.75 fragment promoter sequence was also further investigated for its regulation on expression of the â-glucuronidase (gus) reporter gene in transgenic tobacco. Analysis of gus activities showed that the 0.75 kb flanking fragment of the protease inhibitor gene actually drives the gus reporter gene expression in ovary, pistil and seeds of tobacco. In another case, promoter sequence consisting of 1.3 kb fragment of a 52 flanking region of black gram protease inhibitor gene was isolated by Genome Walker strategy and promoter sequence was analyzed in-silico by PLACE and PlantCARE database.

Overexpression of soybean miR169c confers increased drought stress sensitivity in transgenic Arabidopsis thaliana

The miR169 family, a large-scale microRNA gene family conserved in plants, is involved in stress responses, although how soybean miR169 functions in response to drought stress remains unclear. We show that gma-miR169c exerts a negative regulatory role in the response to drought stress by inhibiting the expression of its target gene, nuclear factor Y–A (NF-YA). A real-time RT-PCR analysis indicated that gma-miR169c is widely expressed in soybean tissues and induced by polyethylene glycol (PEG), high salt, cold stress and abscisic acid (ABA). Histochemical ß-glucuronidase (GUS) staining showed that the gma-miR169c promoter drives GUS reporter gene expression in various transgenic Arabidopsis tissues, and the stress-induced pattern was confirmed in transgenic Arabidopsis and transgenic soybean hairy roots. Arabidopsis overexpressing gma-miR169c is more sensitive to drought stress, with reduced survival, accelerated leaf water loss, and shorter root length than wild-type plants. We identified a precise cleavage site for 10 gma-miR169c targets and found reduced transcript levels of the AtNFYA1 and AtNFYA5 transcription factors in gma-miR169c-overexpressing Arabidopsis and reduced expression of the stress response genes AtRD29A, AtRD22, AtGSTU25 and AtCOR15A. These results indicate that gma-miR169c plays a negative regulatory role in drought stress and is a candidate miRNA for improving plant drought adaptation.

Vacuum infiltration enhances the Agrobacterium-mediated transient transformation for gene functional analysis in persimmon (Diospyros kaki Thunb.)

An Agrobacterium-mediated transient transformation system was established for gene functional analysis in persimmon (Diospyros kaki Thunb.) utilizing sonication followed by vacuum infiltration of seedlings in vitro. The optimal vacuum conditions for agroinfiltration of persimmon were evaluated by monitoring expression of GUS reporter gene. The technique was further applied to evaluate five different genotypes. Among the genotypes examined in vitro, ‘Xiaoguo-tianshi’ (Chinese PCNA type) produced the highest expression levels of both GUS and GFP, and was the most ideal genotype. Furthermore, the developed system was successfully utilized for DkLAR (leucoanthocyanidin reductase) gene transient silencing. DMACA (4- dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay revealed that the proanthocyanidins (PAs) levels was markedly decrease in ‘Xiaoguo-tianshi’ and ‘Mopanshi’ (Chinese PCA type) leaves. These results confirmed that DkLAR plays an important role in the accumulation of PAs in persimmon. In conclusion, based on the Agrobacterium-mediated vacuum infiltration, we developed a reliable and highly efficient transient transformation system that can be used for gene functional analysis through the seedlings in vitro.

Serendipita indica modulates expression of arginine metabolism genes during colonization of Arabidopsis thaliana roots

Abstract Endophytic fungus Serendipita indica belongs to the order Sebacinales. It is an extremely versatile root endophyte colonizing more than 150 different plant species including the model plant Arabidopsis thaliana. Colonization of S. indica triggers enhanced growth, early flowering, increase in seed content, alteration of secondary metabolite pathways and adaption to different biotic and abiotic stresses. This is why this fungus got ample attention by the plant researchers. In this study, we report modulation in expression of argininosuccinate synthase (AS), argininosuccinate lyase (AL) and arginase 2 (ARGAH2) genes in A. thaliana as a result of S. indica colonization. Similarly, expression of GUS reporter gene driven by AS and ARGAH2 promoters in roots was altered. AS, AL and ARGAH2 genes were down-regulated at early stage of root colonization i.e at 3 days post inoculation (dpi), with a subsequent up-regulation at later stages (7 and 14 dpi). Other arginine catabolism genes, Arginine decarboxylase-1 (ADC1), Arginine decarboxylase-2 (ADC2) and Pyrroline-5-carboxylate reductase (P5CR) were strongly up-regulated at later stages of S. indica colonization. Our findings show significant changes in expression of several important genes in arginine metabolic pathway upon S. indica colonization. These changes are very specific and strongly depend on the colonization phase of the fungus. Keywords: AL; ARGAH1; ARGAH2; AS; Arginine metabolism; GUS; Quantitative RT-PCR; Serendipita indica http://dx.doi.org/10.19045/bspab.2018.700203

Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.

TcMYC2a, a Basic Helix-Loop-Helix Transcription Factor, Transduces JA-Signals and Regulates Taxol Biosynthesis in Taxus chinensis.

The multitherapeutic taxol, which can be obtained from Taxus spp., is the most widely used anticancer drug. Taxol biosynthesis is significantly regulated by jasmonate acid (JA), one of the most important endogenous hormones in land plants. Nevertheless, the JA-inducing mechanism remains poorly understood. MYC2 is one of the key regulators of JA signal transfer and the biosynthesis of various secondary metabolites. Here, TcMYC2a was identified to contain a basic helix–loop–helix (bHLH)-leucine zipper domain, a bHLH-MYC_N domain, and a BIF/ACT-like domain. TcMYC2a was also found to bind with TcJAZ3 in yeast, which was a homolog of Arabidopsis JASMONATE ZIM-domain JAZ proteins, indicating that TcMYC2a had a similar function to AtMYC2 of JA signal transduction. TcMYC2a was able to affect the expression of GUS reporter gene by binding with the T/G-box, G-box, and E-box, which were the key cis-elements of TASY and TcERF12/15 promoter. TcMYC2a overexpression also led to significantly increased expression of TASY, tat, dbtnbt, t13h, and t5h genes. Additionally, TcERF15, which played the positive role to regulate tasy gene, was up-regulated by TcMYC2a. All these results revealed that TcMYC2a can regulate taxol biosynthesis either directly or via ERF regulators depending on JA signaling transduction.

Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis.

Auxin regulates nearly all aspects of plant growth and development including cell division, cell elongation and cell differentiation, which are achieved largely by rapid regulation of auxin response genes. However, the functions of a large number of auxin response genes remain uncharacterized. Paclobutrazol Resistance (PRE) proteins are non-DNA binding basic helix-loop-helix transcription factors that have been shown to be involved in gibberellin and brassinosteroid signaling, and light responses in Arabidopsis. Here, we provide molecular and genetic evidence that PRE6, one of the six PRE genes in Arabidopsis, is an auxin response gene, and that PRE6 is involved in the regulation of auxin signaling. By using quantitative RT-PCR, we showed that the expression level of PRE6 was increased in response to exogenously applied IAA. GUS staining results also showed that the expression of GUS reporter gene in the PRE6p:GUS transgenic seedlings was elevated in response to auxin. Phenotypic analysis showed that overexpression of PRE6 in Arabidopsis resulted in auxin-related phenotypes including elongated hypocotyl and primary roots, and reduced number of lateral roots when compared with the Col wild type seedlings, whereas opposite phenotypes were observed in the pre6 mutants. Further analysis showed that PRE6 overexpression plants were hyposensitive, whereas pre6 mutants were hypersensitive to auxin in root and hypocotyl elongation and lateral root formation assays. By using protoplasts transfection, we showed that PRE6 functions as a transcriptional repressor. Consistent with this, the expression of the auxin response reporter DR5:GUS was decreased in PRE6 overexpression lines, but increased in pre6 mutants. When co-transfected into protoplasts, ARF5 and ARF8 activated the expression of the PRE6p:GUS reporter. Chromatin immunoprecipitation assays showed that ARF5 and ARF8 can be recruited to the promoter regions of PRE6. Taken together, these results suggest that PRE6 is an auxin response gene whose expression is directly regulated by ARF5 and ARF8, and that PRE6 is a transcriptional repressor that negatively regulates auxin responses in Arabidopsis.

Promoter Analysis of a Powdery Mildew Induced Vitis vinifera NAC Transcription Factor Gene

Grapevine is an economically important crop throughout the world. However commonly cultivated species Vitis vinifera is susceptible to powdery mildew fungi. The objective of this study was to analyze the promoter of VvNAC36 transcription factor gene, which was induced by powdery mildew infection, as it was shown by previous microarray results. Analysis of VvNAC36 transcription factor promoter allows identifying the important cis-elements, which are responsible for gene regulation during infection. Knowing that the responsible cis-element of the gene is crucial for further study, the role of this gene is in powdery mildew infected grapevine. The study was performed using circumspect methodology. To confirm the presence of transgene in transgenic plants the PCR and glufosinate total herbicide application was used. The transplanted transgenic seedlings were infected with Golovinomyces orontii. To detect the GUS reporter gene expression, histochemical GUS staining and spectrophotometric assays were applied in each deletion variant. Finally the promoter sequence was analyzed by the PLACE (a database for PLAntCisacting Elements) program for identifying cis-elements. The results of the study showed significant difference in mock treated and induced expression in transgenic plants transformed with VvNAC36 promoter segments of different lengths, fused to GUS reporter gene. In basal expression level, the deletion variants 1178 bp and 257 bp differed significantly. Powdery mildew infection significantly increased the expression in all promoter variants, except the two shortest fragments. Based on the promoter motif analysis, it can be concluded, that the GT1-box (GAAAAA) supposed to have crucial role in regulating of VvNAC36 transcription factor gene during powdery mildew infection.

In vitro shoot multiplication and ex vitro rooting of Eustoma grandiflorum by shoot tips using 2,4-D and BAP

An Agrobacterium-mediated transient transformation system was established for gene functional analysis in persimmon (Diospyros kaki Thunb.) utilizing sonication followed by vacuum infiltration of seedlings in vitro. The optimal vacuum conditions for agroinfiltration of persimmon were evaluated by monitoring expression of GUS reporter gene. The technique was further applied to evaluate five different genotypes. Among the genotypes examined in vitro, ‘Xiaoguo-tianshi’ (Chinese PCNA type) produced the highest expression levels of both GUS and GFP, and was the most ideal genotype. Furthermore, the developed system was successfully utilized for DkLAR (leucoanthocyanidin reductase) gene transient silencing. DMACA (4- dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay revealed that the proanthocyanidins (PAs) levels was markedly decrease in ‘Xiaoguo-tianshi’ and ‘Mopanshi’ (Chinese PCA type) leaves. These results confirmed that DkLAR plays an important role in the accumulation of PAs in persimmon. In conclusion, based on the Agrobacterium-mediated vacuum infiltration, we developed a reliable and highly efficient transient transformation system that can be used for gene functional analysis through the seedlings in vitro.

Optimization of Conditions for Genetic Transformation and In Vitro Propagation of Artemisia carvifolia Buch

The excessive attention in the use of plants as medicine is credited to the occurrence of active principles whose pharmacological activities have been investigated. Due to the limited production and very less quantity of these important metabolites present in the plant cells, their genetic engineering and increased in vitro production is the point of focus for many years and can be achieved by in vitro transformation and propagation of desired plant. In the current study, we report transformation protocol for Artemisia carvifolia Buch with Agrobacterium tumefaciens C58C1 harboring β-glucuronidase as reporter and neomycin phosphotransferase as selectable marker gene. We have optimized simple regeneration conditions after transformation involving two different types of explants (leaf and stem) on different media formulations for direct organogenesis and best regeneration response. MS media with 2.5 g/L benzylaminopurine (BAP), 0.25 g/L naphthalene acetic acid (NAA), giving maximum number of shoots was selected. Rooting was obtained on ½ MS medium supplemented with NAA (0.1 mg/L). Transient expression of gus reporter gene was observed in the leaf and stem explants after 2 days of bacterial infection. Artemisia carvifolia Buch can be successfully transformed with Agrobacterium tumefaciens strain C58C1 by using controlled in vitro regeneration conditions. Findings of the current study would be useful for micro propagation and genetic transformation of Artemisia carvifolia Buch in future.

Truncated Ubiquitin 5' Regulatory Region from Erianthus arundinaceus Drives Enhanced Transgene Expression in Heterologous Systems.

Characterization of novel plant gene promoters underpins the development of transgenic crop plants. Here, we report a novel 5' regulatory sequence (Eriubi D7) of the ubiquitin gene from Erianthus arundinaceus, a wild relative of sugarcane resistant to many biotic and abiotic stresses. A 3.2-kb regulatory sequence of ubiquitin gene was isolated through random amplification of genomic ends technique and characterized in rice, tobacco, and sugarcane. In silico analysis revealed that the regulatory sequence contained a promoter region of 1600bp upstream to the transcription start site. Between the promoter and the coding region, two putative introns of 584 and 583bp and two putative non-coding exons of 459 and 37bp were spaced alternatively. To identify the active domains required for gene regulation, 12 truncations/recombinants were made in the regulatory sequence and characterized in heterologous systems. Transformation studies with the recombinant constructs revealed that Eriubi D7, a truncated fragment containing 830bp promoter and the intron I, conferred enhanced GUS reporter gene expression in both monocots and dicots compared to other routinely used promoters such as maize ubi1 and Cauliflower Mosaic Virus 35S. Further analysis confirms that this regulatory sequence is quite distinct from the other reported ubiquitin promoters and was also found to enhance expression of the reporter gene upon wounding. This is the first report on the isolation and characterization of a promoter from a wild sugarcane germplasm and is expected to be useful for development of transgenic crop plants.

Isolation and Characterization of a OsRap2.4A Transcription Factor and its Expression in Arabidopsis for Enhancing High Salt and Drought Tolerance

Dehydration responsive element/C-repeat (DRE/CRT) is a cis-acting element involved in the regulation of abiotic stress-responsive gene expression in higher plants. Using a 50-nucleotide bait containing DRE cis-acting element localized on the downstream mini TATA box of glutamate dehydrogenase-like protein ( JRC2606 ) promoter in yeast one-hybrid screening, we have identified two transcription factors belonging to A6 subgroup of DREB subfamily - OsRap2.4A and OsRap2.4B. Expression of OsRap2.4A was induced by drought, high salt and temperature stress conditions, and also by abscisic acid treatment. Binding assay showed that OsRap2.4A bound specifically to DRE sequence in both in vivo and in vitro experiments. Transient expression of OsRap2.4A in protoplasts revealed that OsRap2.4A functioned as a transcriptional activator and upregulated expression of the GUS re-porter gene. Transgenic Arabidopsis plants expressing OsRap2.4A at low level showed no significant growth re-tardation under normal condition, whereas at high level expression of OsRap2.4A caused an obvious retardation. Expression of OsRap2.4A showed the tolerance against drought and salt stresses in comparison with control. These results suggested that OsRap2.4A gene may be involved in a new regulatory pathway in plant responses to abiotic stresses and it is potentially useful for the transformation into crop plants to improve tolerance against drought and high salt stresses.

Isolation and cloning of fruit-specific E8 promoter from local small fruit tomato (Lycopersicon esculentum L.) and analysis its transient expression

Isolation and characterization of specific promoter is important for efficient expression of recombinant proteins in plants. E8 is a fruit specific promoter with 2.2 kb length. To isolate E8 fruit specific promoter, seeds of local small fruit Sardasht genotype were sown in greenhouse and genomic DNA was extracted from young leaves using CTAB method. The promoter region was amplified using specific primers designed based on conserved regions of the sequences available at NCBI database. The amplified fragments ligated into PTZ57R/T vector and transferred into competence cells of DH5α strain of E. coli for cloning. Plasmid was extracted after confirming the presence of the inserted segments in plasmid based on colony PCR test and enzyme digestion. The extracted plasmids were used for sequencing the fragments. Sequence analysis revealed successful isolation of E8 promoter segment. To analyze the activity promoter in tomato tissues, the CaMV35S promoter in pBI121 binary vector replaced by E8 promoter and transferred to GV3101 strain of Agrobacterium. Transient expression was assessed using gus gene under E8 promoter with aim of transgenic Agrobacterium agro-infiltrated into fruit tissue. Presence of indigo color indicated successful expression of gus reporter gene under E8 promoter at the cells receipt the gene construct.

Rapid, Efficient and High-Performance Protocol for <i>Agrobacterium rhizogenes</i>-Mediated Hairy Root Transformation of the Common Bean <i>Phaseolus vulgaris</i>

A rapid, efficient and high-performance transformation protocol employing Agrobacterium rhizogenes was developed for the common bean Phaseolus vulgaris. In this study, we examined competencies of various protocols to induce and explants that respond to hairy root transformation in bean plants. Utilizing young seedlings with severed radicles/hypocotyls, we developed a highly efficient procedure for achieving hairy root transformation frequencies as high as 100% as visualized by GUS reporter gene expression system. Transgenic hairy roots in these young composite plants were susceptible to nodulation by rhizobia, and form an excellent system for high throughput genomic analysis to study root biology and endosymbiosis in common bean.

Expression patterns and regulation of SlCRF3 and SlCRF5 in response to cytokinin and abiotic stresses in tomato (Solanum lycopersicum)

Cytokinin is an influential hormone involved in numerous aspects of plant growth and development. A group of transcription factors–cytokinin response factors (CRFs) has been included as a side branch to cytokinin signaling pathway which also constitute a subset of the AP2/ERF family of transcription factor proteins. This study examined the expression patterns of two transcription factor genes SlCRF3 and SlCRF5 in tomato (Solanum lycopersicum) to determine their regulation in response to cytokinin and a variety of abiotic stress conditions. Analyses conducted during different developmental stages by RT-PCR or GUS reporter gene expression revealed that these genes are differentially expressed in vegetative and reproductive organs. qRT-PCR experiments were also performed to study regulation by the hormone cytokinin and abiotic stress conditions such as flooding, drought, osmotic, oxidative, and temperature. These showed that SlCRF3 and SlCRF5 have different patterns of regulation in leaf, stem, and roots with SlCRF5 showing greater induction in leaf or root tissue compared to SlCRF3 in most cases. Additionally, knockdown analysis for SlCRF5 revealed defects across development including leaf morphology, primary root growth, and lateral root formation. Together, these findings indicate that SlCRF3 and SlCRF5 are potential regulators of tomato developmental processes associated with cytokinin or abiotic stresses.

Characterization of the Promoter of Artemisia annua Amorpha-4,11-diene Synthase (ADS) Gene Using Homologous and Heterologous Expression as well as Deletion Analysis

Amorpha-4,11-diene synthase (ADS) is the first key enzyme of artemisinin biosynthetic pathway in Artemisia annua L. In this study, the promoter region of the ADS gene has been cloned and used to demonstrate the expression of GUS reporter gene in both glandular trichomes of A. annua and non-glandular trichomes of Arabidopsis thaliana following homologous and heterologous expression of ADS promoter–GUS fusion. Subsequently, 5′ sequential deletion analysis of the ADS promoter revealed that a short sequence, −350 upstream of the transcription start site, was sufficient for trichome-specific expression in A. thaliana and that the region from −350 to −300 contained essential elements for this observed specificity. However, frequencies of transgenic A. thaliana plants displaying trichome-specific expressions varied between different lines, and all the lines with deleted fragments of the ADS promoter showed lower frequencies than the line with full-length ADS promoter. Most lines with deleted ADS promoter–GUS fusions showed GUS expressions in the guard cells of stomata as well, which was not observed in A. thaliana plants transformed with the full-length ADS promoter. GUS activities varied among different transgenic lines as well, both in transiently transformed Nicotiana benthamiana and stably transformed A. thaliana, with promoter–deletion lines exhibiting higher GUS activities than the full-length ADS promoter line.

PpERF3b, a transcriptional repressor from peach, contributes to disease susceptibility and side branching in EAR-dependent and -independent fashions

Peach ERF3b is a potent transcriptional repressor for defense-related genes even in the presence of similar levels of transcriptional activators and can interfere with plant development through pathways independent of the EAR motif. Ethylene response factors (ERFs) are a major group of plant transcription factors with either activation or repression capabilities on gene transcription. Repressor-type ERFs are characterised by an intrinsic motif, namely the ERF-associated amphiphilic repression motif (EAR). Here we report the identification of three genes from peach (Prunus persica), PpERF12, PpERF3a and PpERF3b, encoding for ERF repressors. The transcription kinetics of these genes was investigated by qRT-PCR after inoculation of peach leaves with Xanthomonas campestris pv. pruni. All three genes showed higher induction in the susceptible 'BabyGold 5', than in the resistant 'Venture' peach varieties suggesting a negative role for these genes in disease resistance. The functional potency of PpERF3b has been confirmed in vivo by its ability to repress the expression of GUS-reporter gene. To better understand the functional role of PpERF3b, the full-length and the EAR-truncated (PpERF3b∆EAR) genes were overexpressed in tobacco (Nicotiana tabacum). Both transgenic plants (PpERF3b and PpERF3b∆EAR) uniformly exhibited precocious side branching, which suggests the interference of PpERF3b with auxin-mediated dormancy of lateral shoots. Consistent with that the expression of auxin-response factors (Nt-ARF1, Nt-ARF6 and Nt-ARF8) was significantly downregulated in transgenic plants compared to the wild type (WT). Although side branching was independent of EAR motif, the response of transgenic plants to inoculation by Pseudomonas syringae pv. tabaci was EAR dependent. Transgenic plants overexpressing PpERF3b∆EAR showed less disease symptoms than those overexpressing the full-length gene or WT plants. Resistance of PpERF3b∆EAR plants was associated with enhanced induction of pathogenesis-related (PR) genes. Our results indicate that repressor-type ERFs might act through pathways that are dependent or independent of the EAR motif.